Gram-negative flagellin is an extraordinarily potent activator of innate and adaptive immune responses. Our work as well as that of others has established that flagellin is a potent adjuvant in mice. We have also found that flagellin is a potent adjuvant in non-human primates. Flagellin possesses features that make it an excellent adjuvant including: potency;mucosally active;effectiveness in flagellin immune hosts;ease of production in bacteria;and 5) the ability to insert antigen sequences in the hypervariable region of the protein without loss of flagellin bioactivity. We have clearly established that flagellin is a highly effective mucosal adjuvant in the development of a strong, specific antibody response against Y. pestis antigens in mice and non-human primates and also promotes protection against a lethal respiratory challenge with Y. pestis. Since flagellin possesses tremendous potential as a mucosal adjuvant for use in humans, we propose the following aims that will advance the development of flagellin for human use: Specific Aim 1. To determine the efficacy of a flagellin/Y. pestis F1 and V fusion protein as a protective mucosal vaccine. We have generated a recombinant protein encoding flagellin and both the F1 and V antigens of Y. pestis and have established that this fusion protein retains flagellin biological activity. We propose to determine if the fusion protein promotes a robust anti-F1 and V IgG response in mice and cynomolgus monkeys and protection in mice against respiratory challenge with Y. pestis CO92. Specific Aim 2. To design and implement a Good Manufacturing Practice (GMP) compatible production process for a flagellin/Y. pestis vaccine that can be readily scaled for pre-clinical, clinical and ultimately commercial use. Specific Aim 3. To conduct GLP toxicology studies with the flagellin/Y. pestis vaccine. Once the process has been optimized, an engineering lot will be prepared to study its safety and immunogenicity under GLP conditions in a mouse model. This lot will also be used for characterization studies and to initiate formal stability studies in preparation for clinical manufacture. Specific Aim 4. To produce a flagellin/Y. pestis vaccine under Good Manufacturing Practices (GMP) conditions. The protocol developed in Specific Aim 2 will be used to produce gram quantities of the vaccine under GMP conditions. The completion of these aims will provide a very strong foundation for the clinical evaluation of the mucosal flagellin/Y. pestis vaccine in humans.